1. Field of the Invention
This invention relates to the field of tissue engineering, and in particular to a method of processing animal tissue including, particularly, fetal or neo-natal tissue to produce a biopolymer scaffold material named EB Matrix (xe2x80x9cEBMxe2x80x9d). EBM has broad applications for tissue repair and regeneration. It can serve as a remodelable scaffold for repair or replacement of human tissues and organs. It can be enriched with signaling molecules and cells before implantation, or with or without signaling molecules, it can attract host vessels and vascular cells as well as host parenchymal cells and immune systems cells to populate it after implantation. Also, it can serve as a delivery device for signaling factors, cells or drugs.
2. Description of the Related Art
Rebuilding the human body is a significant industry. Human tissue banks and synthetic polymers do not meet the need for repair or replacement of body parts. High on the list of alternative sources of material used to meet this need are animal tissues prepared in new ways that reduce their immunogenicity and maximize their usefulness and efficacy.
In the field of tissue engineering, the following three components are used alone or in combination to repair or create new tissue and organ substitutes. 1) scaffolds made of naturally-occurring polymers (e.g. collagens), man-made polymers, (e.g. PTFE, Dacron, PET or polyethylene) or self-degrading, man-made polymers (e.g. PLA or PGA); 2) signaling molecules that give developmental instructions to cells; and 3) cells having specific or multiple tissue building potential, often referred to as xe2x80x9cstem cellsxe2x80x9d. Here we describe biopolymer matrices, produced by novel methods, from animal tissues including fetal and neo-natal tissues to be used as tissue engineering scaffolds.
Man-made implant materials such as synthetic polymers, plastics, and surface-coated metals may have different degrees of immunogenicity and suffer from significant limitations that prohibit their broad applications. A major limitation is that cells cannot remodel them after implantation. They are highly susceptible to microbial infection, and some undergo calcification. Synthetic vascular conduits have a high incidence of occlusion after peripheral vascular bypass procedures.
There is a long history of the use of biopolymer matrices made from processed human or animal tissues. Several methods of preserving collagen-based matrices from animal tissues have been developed (U.S. Pat. Nos. 4,801,299, 5,336,616, 5,756,678, 5,916,265 and 5,997,895). All the methods include a chemical step that either kills or eliminates cells. Since tissues from post-natal animals or humans are the principal materials processed, a fixation step using glutaraldehyde or a similar agent may be used to mask antigenic determinants, eliminate the microbial burden and increase strength. However, aldehydic processing effectively destroys any biological activity, such as cell binding sites, associated with the original tissue and greatly reduces or eliminates the ability of cells to attach to it. It also eliminates binding sites for cell-synthesized products which attach to cells or to intermediates able to bind to cells and cell products that make up the extracellular matrix by cells.
Collagen-based devices that are animal-derived and fixed with glutaraldehyde or a similar agent can not be remodeled since they are highly resistant to metalloproteinase enzymes. Glutaraldehyde-treated devices are known to undergo gradual calcification. Heart valves made from fixed animal tissues can require replacement in 5-7 years or sooner due to calcification. The methods suggested in U.S. Pat. Nos. 4,801,299 and 5,916,265 include the use of glutaraldehyde or a similar agent for the fixation of tissue derived from a post-natal animal source. The resulting products can not be faithfully remodeled.
While detergents or sodium hydroxide may be used to process post-natal animal tissue (U.S. Pat. Nos. 4,801,299, 5,336,616, 5,756,678, 5,916,265, 5,997,895), they have not been used to process fetal or neo-natal animal tissue. For example, U.S. Pat. No. 5,997,895, filed on Apr. 30, 1998, provides a certified collagen dural substitute derived from post-natal animal tissue that undergoes an alkaline/salt treatment involving sodium hydroxide and sodium sulfate (preferably in an aqueous solution of 5% sodium hydroxide and 20% sodium sulfate). A method for processing collagen containing materials which uses 1.0 N sodium hydroxide was disclosed in a journal article in 1989 by Diringer H. and Braig H. R. (Diringer H. and Braig H. R., 1989, Infectivity of unconventional viruses in dura mater. The Lancet, 439-440). This reference was cited in the FDA""s Guide for 510(k) Review of Process Dura Mater (1990, 2).
The product of this invention, EBM, is unlike the other products cited above which in general are not bioremodelable. EBM is processed in a way that preserves its binding sites for cells and cell-secreted products that make up the extracellular matrix surrounding cells that occupy the scaffold. EBM is also distinguished by the fact that undesirable tissue components, such as DNA, are expressed mechanically from the tissue and that delipidyzing organic solvents are used to reduce the presence of cell and nuclear membranes. EBM does not calcify, making it safe for use in the human body for repair of soft tissues. In addition to its use for soft tissues, EBM can be used as a scaffold for bone repair if treated with an appropriate growth factor, if seeded with bone precursor cells or if occupied by bone forming cells when implanted.
EBM can be used as a tissue-building component with or without cells or signaling complexes for creating human body replacements. It can be used after the addition of signaling molecules, which will further promote tissue repair. It can also be implanted after stem or differentiated cells are seeded into or onto it.
By processing animal tissue, including fetal or neo-natal tissue, by the method embodying the invention, tissue strength is preserved without reducing its intrinsic biological properties or compromising the ability of cells that occupy the tissue to remodel it. In addition to chemical processing, a step of mechanically expressing undesirable tissue components from the tissue is a significant innovation. Additionally, the uniqueness of this invention is that it includes the use of fetal or neo-natal tissue that, depending on age, is much less antigenic than adult tissue. The present invention overcomes the difficulties inherent in the approach to animal tissue use based on glutaraldehyde treatment.
For convenience, certain terms used in the specification, examples, and appended claims are collected here alphabetically.
The term xe2x80x9cbioremodelablexe2x80x9d or xe2x80x9cbioremodelabilityxe2x80x9d refers to a material that lends itself to the breakdown by cells that occupy it and use it as a template for creating a replacement made up mainly of newly synthesized components secreted by the cells.
The terms xe2x80x9cdelipidizingxe2x80x9d or xe2x80x9cdelipidizedxe2x80x9d refers to the removal of lipids from the tissue.
The term xe2x80x9cDHTxe2x80x9d refers to a dehydrothermal process, wherein the tissue is cross-linked and dehydrated at a high temperature.
The term xe2x80x9cdrapabilityxe2x80x9d includes the capacity of the material to mold to irregular, curved surfaces or surfaces of other geometries.
The terms xe2x80x9cinactivatingxe2x80x9d or inactivatedxe2x80x9d refers to the reduction of the concentrations of infective agents (e.g. bacteria, molds, viruses and prions) by 4, 6 or 8 logs consistent with the requirements needed to insure against infectivity.
The phrase xe2x80x9cmechanically expressingxe2x80x9d refers to mechanically applying pressure to express undesirable components from the tissue. With the aid of appropriate solvents, unwanted components from the product that are potentially antigenic, such as DNA, RNA or other molecules released by reagents, such as NaOH, are removed.
The term xe2x80x9csuturablexe2x80x9d includes the ability to suture the material, wherein the material offers the required resistance to suture pull-out.
This invention involves preserving a naturally occurring, biopolymer-based matrix (EBM) from animal tissue, particularly fetal or neo-natal tissue. It provides a method of producing EBM, including the following steps: (1) removing the tissue from its source; (2) optionally extracting growth and differentiation factors from the tissue; (3) inactivating infective agents of the tissue; (4) mechanically expressing undesirable components from the tissue; (5) washing the tissue for removal of chemical residues; (6) optionally drying; and (7) optionally cross-linking the tissue after chemical and mechanical treatment.
In the preferred embodiment, porcine or bovine tissues, including fetal and neo-natal tissues, are used. Preferably, for example the fetal bovine tissue source is between 10 weeks of age and newborn age. As an example, fetal bovine skin is flash frozen and stored. Other source material include blood vessels, other tubular structures, internal organs including the bladder, tendons, ligaments, cartilage, membranes such as the kidney capsule or diaphragm, or hard tissues such as cartilage or bone. After thawing, the tissue or organ is kept cold and chilled in an ice bath at a temperature between xe2x88x924xc2x0 C. and 10xc2x0 C. In a preferred embodiment, a salted ice bath is used to chill the tissue to a temperature below 0xc2x0 C. The tissue adhering to the underside of the skin for example is mechanically removed.
Whereas, certain desirable naturally-occurring components of tissues, particularly fetal and neo-natal tissues, may be lost because of the harsh chemicals used for viral and prion inactivation and removal of unwanted structures and chemical components, some at high temperatures, desirable components such as growth and differentiation factors may be extracted from the skin or other tissue before the bleach and sodium hyrdroxide treatments for the purpose of viral and prion inactivation and for removal of unwanted structural and chemical components. In an alternative embodiment, growth and differentiation factors are extracted from the tissue by methods disclosed in U.S. Application No. 60/251,125, filed on Dec. 4, 2000, herein incorporated by reference (e.g. buffer, enzyme or acid extraction). The extracted growth and differentiation factors, being in solution, are treated much more mildly with the agents used for viral and prion inactivation, such as with 1 N sodium hydroxide for 4 hrs. on a shaker on ice. After treatment, the extracted growth and differentiation factors are returned to the skin or other tissue being processed which readily absorbs them.
In the preferred embodiment, the skin undergoes microbial, fungal, viral and prion inactivation, beginning with a treatment with bleach. The bleach is at a concentration of between 0.05% and 5%, and the time of treatment can vary between 1 minute and 5 hours. This step can also be done after the sodium hydroxide step described below. All solutions are chilled with ice or salted ice to a temperature between xe2x88x924xc2x0 C. and 10xc2x0 C.
In the preferred embodiment, the tissue is washed extensively with water or physiological buffers (e.g. Tris-, HEPES, PBS buffer) to remove any residual bleach. The tissue is treated further with sodium hydroxide or potassium hydroxide at a concentration of between 0.1 N and 10 N for between 10 minutes and 2 hours. This treatment also inactivates infective agents of the tissue (e.g. bacteria, molds, viruses and prions). The container and all solutions are chilled with ice or salted ice to a temperature between xe2x88x924xc2x0 C. and 10xc2x0 C. The container is subsequently placed on a shaker.
In the preferred embodiment, unwanted components from the tissue (e.g. DNA, RNA or other molecules released by reagents such as NaOH) are mechanically expressed by means of repeated applications of pressure using a flat blade (like a putty knife) and/or roller(s). The steps of mechanically expressing material dissociated from the tissue chemically can be carried out by a machine as well, through an operation similar to that used manually.
Any organic solvent as well as mixtures of them suitable for dissolving lipids may be used for removal of lipid materials (e.g. chloroform, acetone, ether, alcohols and their mixtures). In the preferred embodiment, the tissue is delipidized in a chloroform and ethanol mixture (1:1 concentration ratio) for between 5 minutes and 5 hours followed by washes in 70% ethanol and water, or the foregoing solvents can be used seriatim with the ethanol being at a concentration of 70% for similar periods of time applied after the chloroform step.
In the preferred embodiment, the tissue is subjected to extensive washing with distilled water or buffers until the chemical residue is removed. The final product that is designated as EBM, is stored in distilled water or buffers, or dried.
In an alternative embodiment, EBM can be cross-linked with genipin or DHT. If DHT is used, the tissue undergoes dehydration at a high temperature.
In an alternative embodiment, EBM can be freeze-dried by rapidly freezing the tissue and then dehydrating it in a high vacuum. Freeze-drying increases the porosity and flexibility of the tissue.